ABSTRACT Under the support of the previous grant, we tested the hypothesis that PKC?, an important stress regulator, contributes to smooth muscle cell depletion and vascular inflammation during aneurysm development. Using a combination of genetic, molecular, and pharmacological approaches, we demonstrated the essential role of PKC? in regulating vascular smooth muscle cell (SMC) apoptosis in aneurysm. Unexpectedly, we also discovered a novel connection between PKC? and necroptosis, a form of programmed necrosis first described a decade ago. Receptor-interacting protein kinase 3 (RIP3) and its partner RIP1 are among the few identified mediators of necroptosis, underscoring the significance of positioning PKC? within this pathway. We demonstrated that in human aneurysm tissue, SMC levels of both PKC? and RIP3 was upregulated. In mice, gene deletion of either PKC? (Prkcd-/-) or RIP3 (Rip3-/-) produced an aneurysm-resistant phenotype associated with preserved SMCs and diminished inflammation. In preliminary studies, we showed that PKC? regulates Rip3 gene transcription in aortic SMCs, a novel finding highly expected to advance RIP3 biology. The robust aneurysm-protective phenotype of Rip3-/- and Rip3+/- motivated a chemical library screening which led to the discovery of a class of potent and safe RIP3 inhibitors. In this renewal application, we hypothesize that PKC?- STAT3 signaling is a determinant of SMC Rip3 expression and that inhibition of RIP3 may attenuate growth of pre-existing aneurysms. Our objectives for the next 5 years include 1) to determine the molecular mechanism underlying PKC?-mediated Rip3 gene expression in aneurysmal aortic wall, 2) to establish a more comprehensive necroptosis signaling network in aortic SMCs, and 3) to use the new RIP3 inhibitor to block disease progression of pre-existing aneurysms in mice. Two independent specific aims are proposed. In Specific Aim 1, we plan to prove the critical role of STAT3 in PKC?'s regulation of Rip3 by rescuing Prkcd-/- SMCs with a constitutively active STAT3. Next, we will determine whether STAT3 Serine727, a less studied regulatory mechanism, is phosphorylated in aneurysm tissue via a PKC?-dependent mechanism. Mechanistically, we postulate that STAT3 regulates Rip3 gene transcription through a downstream cis-element. We will test this hypothesis using cutting edge technologies such as Chip-sequencing and CRISPR/Cas9-mediated gene editing. Specific Aim 2 is both basic and translational, with a goal to address the current knowledge gaps in necroptosis biology and to advance therapeutic development for aneurysm. We will utilize the new RIP3 inhibitors to study necroptosis, addressing the relationship between RIP3 kinase inhibition and apoptosis induction and identifying new RIP3 substrates specific to SMCs. In addition to hypothesis-driven approaches, we will employ phospho- proteomics to unbiasedly identify new components of the necroptosis pathway unique to SMCs. Finally, we will prove in vivo that the new RIP3 inhibitors can reverse SMC depletion, tissue destruction and inflammation when administered to mice with pre-existing aneurysmal dilations. Information produced by the proposed studies is likely to have a high impact on the understanding of programmed necrosis as well as aneurysm therapeutic development.